The proposed research is a continuation of our investigations into the mechanism by which an "active oxygen" species is created from molecular oxygen and inserted into substrate molecules. In these studies we will utilize the specific steroid 11B-hydroxylase multienzyme system from bovine adrenocortical mitochondria the characteristics of which have been systematically worked out in our laboratory. This system will in turn serve as a suitable model for other mammalian mixed function oxidase systems acting on drugs and a large variety of other xenobiotic agents. The proposed research centers around the following areas: 1. Further improvements in the methods of preparation and purificatton of the heme protein P-450 and the auxiliary enzyme components, iron sulfur protein and flavoprotein, from adrenocortical mitochondria. 2. Detailed characterization of the chemical and physical properties of the heme protein P-450 in its various forms which it assumes in the reaction cycle of the steroid hydroxylation reaction which was previously established. Efforts will be made to translate these observations into structural information and to provide some insight into the electronic structure of the participating forms of the heme protein. 3. Investigations into the pathways of electron flow during the catalytic cycle of steroid hydroxylation, using the isolated and purified enzyme components in solution and after incorporation into artificial membrane systems (detergent micelles and/or phospholipids). Special efforts will be made to investigate the mechanism by which the second reducing equivalent of the hydroxylation reaction is introduced specifically by the adrenal iron sulfur protein. Optical absorption measurements and EPR spectroscopy will be used throughout in combination with suitable chemical procedures. Kinetic measurements will be used to help elucidate the underlying mechanisms.